Other substrates lack PY motifs and instead rely on interactions with adaptor proteins that recruit the Nedd4 E3 ligase to them, exemplified by a family of arrestin-related trafficking adaptors (ARTs) that bridge the association between substrates and Rsp5 for ubiquitination ( Lin et al., 2008). Tryptophan-tryptophan (WW) domains of Nedd4 family E3 ligases bind to substrate proteins via interaction with PY motifs containing a consensus sequence P/L-P-x-Y ( Rotin and Kumar, 2009 Schild et al., 1996). The Nedd4/Rsp5 family E3 ligases are responsible for membrane protein ubiquitination, required for endocytosis and lysosome-dependent protein degradation. Together, our study uncovers a novel ubiquitination modification implemented by Rsp5 adaptor proteins, underscoring the regulatory mechanism of how adaptor proteins control the recruitment, and activity of Rsp5 for the turnover of membrane proteins. Furthermore, we discover that the homologous to E6AP C-terminus (HECT) domain exosite protects the K63-linked di-ubiquitin on the adaptors from cleavage by the deubiquitination enzyme Ubp2. In agreement with these observations, we find that di-ubiquitin strengthens the interaction between the pombe orthologs of Rsp5 and Art1, Pub1, and Any1. This modification enhances the plasma membrane recruitment of Rsp5 by Art1 or Art5 upon substrate induction, required for cargo protein ubiquitination. Remarkably, we demonstrate that Art1, Art4, and Art5 undergo K63-linked di-ubiquitination by Rsp5. Several arrestin-related trafficking adaptors (ARTs) of Rsp5 were self-ubiquitinated for activation, but the regulation mechanism remains elusive. Nedd4/Rsp5 family E3 ligases mediate numerous cellular processes, many of which require the E3 ligase to interact with PY motif containing adaptor proteins.
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